Tenecteplase (TNK-tPA) is a recombinant glycoprotein of serine protease family with six amino acids substitution in the native human tissue plasminogen activator (t-PA) with 17 disulphide bridges and having molecular weight of ˜67 kDa. In the development of TNK-tPA, the modifications made in native t-PA includes substitution of threonine 103 with asparagine, substitution of asparagine 117 with glutamine both within the kringle 1 domain, and the substitution of lysine, histidine and two arginine with tetra-alanine amino acids at 296-299 positions in the protease domain to make the resulting protein highly fibrin specific with longer plasma half life and 80% decreased susceptibility to degradation by plasminogen activator inhibitor-1 (PAI-1) compared to native t-PA.
The TNK in TNK-TPA refers to the sites of the t-PA molecule that have been modified i.e. T103; Ni 17; and KHRR 296-299. The aforementioned modifications of TNK-tPA renders its use as an improved therapeutic agent for the treatment of acute myocardial infarction which has better therapeutic compliance because the greater fibrin specificity allows for faster and complete clot lysis with decreased bleeding complications and the long life-span permits a single bolus dose with less systemic fibrinolysis and lesser bleeding complications from the previous clot buster drugs.
The mechanism of TNK-tPA is initiated on binding of TNK-tPA to the fibrin component of the thrombus (blood clot) which selectively converts inactive plasminogen into plasmin and consequently the resultant plasmin degrades the matrix of thrombus in occluded artery while conserving fibrinogen and minimizing systemic plasminogen activation due to its highly specific nature.
The benefits of TNK-tPA seen in myocardial infarction patients and the encouraging results from animal studies in the context of Acute Ischemic Stroke (AIS), suggested that TNK-tPA might prove to be a safer and more effective therapy than alteplase, the only drug approved by USFDA for AIS. Over the past few years, several clinical trials evaluated the use of TNK-tPA in AIS and proved that TNK-tPA has a better pharmacological profile than alteplase and also suggested that it could be an effective and safe therapeutic option in treating AIS in patients reporting within 4.5 h after symptom onset. Recently, TNK-tPA has been considered for the treatment of patients with pulmonary embolism and several clinical trials showed promising outcomes. A large number of clinical trials are still being conducted to assess the complete conclusive picture of TNK-tPA in several indications.
In the last few years, development and manufacturing of recombinant glycoproteins was carried out by batch, fed batch, semi-fed batch and perfusion bioreactors processes and for purification of these proteins adsorption and ion exchange chromatography were majorly employed.
For t-PA and its variants certain purification protocols are known in prior art e.g. purification by immuno affinity (anti-tPA goat polyclonal antibody), ion exchange, ethanol precipitation, reverse phase chromatography, chromatography on silica or anion exchange, such as diethylamino ethyl, ammonium sulphate precipitation, sephadex-G-75 etc.
Some of the approaches for the purification of TNK t-PA is listed in prior art includes WO 2011/015922, sets out a purification process where series of ion exchange chromatography steps, immunoaffinity chromatography and ultrafiltration/diafiltration steps are used for purification of TNK-tPA. WO 2012/066569 A, sets out a purification process primarily drawn to the use of hydrophobic interaction chromatography.
The immune affinity chromatography used in the prior art is not suitable technique for commercial manufacturing of TNK-EPA. Not only it could raise lot of regulatory concerns but the cost of immune affinity chromatography media is also very high compared to conventional chromatography matrices owing to their use of monoclonal antibodies for preparation. Hydrophobic interaction chromatography described in certain prior art for TNK-tPA purification uses isopropyl alcohol (IPA) in the process which is an organic solvent and known for inducing aggregation and denaturation of proteins and may be considered as one of the disadvantages of the prior art. TNK-tPA is a highly unstable molecule and hence use of IPA in the purification process should be avoided as it may lead to the denaturation of the protein. In addition, the large volume usage of IPA at commercial scale, would require recycling of IPA which regain demands additional energy consumption and extra investment such as solvent recovery unit.
Therefore, none of the aforementioned processes are capable of providing an efficient, scalable and robust purification solution, which could consistently produce TNK-tPA drug substance at commercial scale, meeting all the required specifications.
Hence, there is a need for an effective and commercially viable process for purification of TNK-tPA.